A coumarin with an unusual structure from Cuphea ignea, its cytotoxicity and antioxidant activities.

Phenolic metabolite profiling using two dimensional paper chromatographic analysis (2 DPC) was used for assaying the complex mixture of phenolics of an aqueous ethanol aerial part extract of Cuphea ignea (Lytheraceae). A coumarin with a rare structure, namely, 7-hydroxy 3-methoxy coumarin 5-O-ß-glucopyranoside was isolated from the investigated extract. The structure was elucidated by conventional methods and spectral analysis, including one and two dimensional NMR (1D and 2D NMR), as well as by interpretation of the spectra obtained by high resolution electrospray ionization mass technique (HRESIMS). The rare coumarin significantly inhibited reactive oxygen species production with an ED50 value of 6.31±1.64 µg/ml and 5.78±0.66 µg/ml as determined by the the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the oxygen radical absorption capacity (ORAC) assay respectively. The isolated coumarin presented a cytotoxic activity assessed by using the neutral red assay (NRU) against lung cancer cell line (H23) with IC50 of 40.38±2.75 µg/ml.

Phenolic profiling of an extract from Eugenia jambos L. (Alston)--the structure of three flavonoid glycosides--antioxidant and cytotoxic activities.

Phenolic metabolite profiling and identification using high performance liquid chromatography (HPLC) coupled to high resolution accurate mass spectrometry (HR-ESI-MS) with detection of negative ions was used for assaying the complex mixture of phenolics of an aqueous ethanol leaf extract of Eugeniajambos L. (Myrtaceae). Eight known polyphenolics were tentatively identified, and, in addition, three hitherto unknown flavonol-O-glycosides were detected in the extract. These unknowns were taken as the targets and isolated by means of consecutive polyamide S6, MCI gel and repeated Sephadex LH-20 column fractionation. The isolation and purification were monitored by HPLC/ESI-MS. The isolates were subsequently identified as quercetin 3-O-xylosyl-(1"' --> 2")-O-xyloside, myricetin 7-methylether 3-O-xylosyl-(1"' --> 2")-rhamnoside and myricetin 3',5'-dimethyl ether 3-O-xylosyl-(1"'-->* 2")-O-rhamnoside. All known metabolites were also separated by applying the same chromatographic techniques. ESI-MS, ¹H and ¹³C NMR spectra were then recorded, completely interpreted and confirmed by HR-ESI-MS and 2D NMR spectroscopy. In order to get information about biological activities of E. jambos the extract was tested for radical scavenging activity by DPPH and ORAC assay. In addition, its cytotoxicity was assessed by the neutral red assay against non-tumorigenic HaCaT keratinocytes and the human bladder carcinoma cell line 5637.

Ethnobotany, chemical constituents and biological activities of the flowers of Hydnora abyssinica A.Br. (Hydnoraceae).

Hydnora abyssinica A.Br. (Hydnoraceae), a holoparasitic herb, is for the first time recorded for Abyan governorate of South Yemen. Flowers of this species were studied for their ethnobotanical, biological and chemical properties for the first time. In South Yemen, they are traditionally used as wild food and to cure stomach diseases, gastric ulcer and cancer. Phytochemical analysis of the extracts showed the presence of terpenes, tannins, phenols, and flavonoids. The volatile components of the air-dried powdered flowers were identified using a static headspace GC/MS analysis as acetic acid, ethyl acetate, sabinene, α-terpinene, (+)-D-limonene and γ-terpinene. These volatile compounds that characterize the odor and taste of the flowers were detected for the first time in a species of the family Hydnoraceae. The flowers were extracted by n-hexane, dichlormethane, ethyl acetate, ethanol, methanol and water. With exception of the water extract all extracts demonstrated activities against Gram-positive bacteria as well as remarkable radical scavenging activities in DPPH assay. Ethyl acetate, methanol and water extracts exhibited good antifungal activities. The cytotoxic activity of the extracts against FL cells, measured in neutral red assay, was only weak (IC50 > 500 µg/mL). The results justify the traditional use of the flowers of Hydnora abyssinica in South Yemen.

Beta vulgaris subspecies cicla var. flavescens (Swiss chard): flavonoids, hepatoprotective and hypolipidemic activities.

The novel flavonoids, 2",2"'-di-O-α-rhamnopyranosyl-vicenin II, a di-C-glycosyl flavone, and herbacetin 3-O-ß-xylopyranosyl- (1"' --> 2")-O-ß-glucopyranoside, were isolated from the leaves of Beta vulgaris subspecies cicla L. var. flavescens, an edible plant which is consumed in the Mediterranean areas, additional to the known flavonoids, 6-C-glucosyl isoscutellarein, vitexin-(1"' --> 2")-O-ß-xylopyranosyl, vitexin-(1'" --> 2")-O-α-rhamnopyranosyI and vitexin. All metabolites were established by conventional methods of analysis and their structures were confirmed by spectroscopic analysis, including 1 D and 2D-NMR and by HR-ESIMS, as well. The extract of the plant leaves shows hepatoprotective effects in rats intoxicated by administration of acetaminophen and exhibits hypolipidemic activity in rats with high-fat-diet induced hypercholesterolemia. The evaluation was done through measuring the liver function enzymes (aspartate and alanine aminotransferases and alkaline phosphatase, the lipid profile (total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and triglycerides) and histopathological analysis of liver slides.

Phenolics from Caesalpinia ferrea Mart.: antioxidant, cytotoxic and hypolipidemic activity.

Nine phenolics were isolated from the aqueous ethanol extract of the leaves of Caesalpinia ferrea. The isolates were characterized for the first time from that plant. The structures of all isolates (1-9) were elucidated by conventional methods, spectroscopic analysis, including 1 D and 2D NMR, and by HR-ESIMS as well. The antioxidant capacities using the ORAC method and the cytotoxic activity using the neutral red assay (NRU) for that extract and three major isolates have been evaluated. In addition, the hypolipidemic activity (in vitro and in vivo) of the extract has been assessed.

Biological activities of selected basidiomycetes from Yemen.

In a previous paper we demonstrated the results of biological screening of Yemeni basidiomycetes. The present study was aimed to investigate the antimicrobial and the antioxidant activity of further basidiomycetes collected in Yemen. Dichloromethane, methanol and aqueous extracts of the fruiting bodies of 25 species were screened in vitro for their antibacterial activities against three Gram-positive bacteria (Staphyloccocus aureus, Bacillus subtilis, Micrococcus flavus) and two Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), against six human fungal pathogens (Candida albicans, Candida krusei, Aspergillus fumigatus, Mucor sp., Microsporum gypseum, Trichophyton mentagrophytes) and against one non human pathogenic fungus (Candida maltosa). The results indicated that 75 extracts exhibited activity against one or more of the bacteria. The methanol extracts of Agaricus cf. bernardii, Agrocybe pediades, Chlorophyllum molybdites, Coriolopsis polyzona, Ganoderma xylonoides, Pycnoporus sanguineus, Trametes lactinea and Trametes cingulata showed activity against all tested bacteria. The highest antibacterial activity was exhibited by methanol extracts from Chlorophyllum molybdites, Ganoderma xylonoides and Trametes cingulata and Agaricus cf. bernardii, Agrocybe pediades, Coriolopsis polyzona, Pycnoporus sanguineus and Trametes lactinea. The methanol extracts of Chlorophyllum molybdites, Ganoderma xylonoides and Pycnoporus sanguineus showed considerable antifungal activities against the tested fungal strains. Strong antioxidative effects employing the DPPH assay were exhibited by methanol extracts from Chlorophyllum molybdites, Ganoderma xylonoides, Hexagonia velutina, Pycnoporus sanguineus, Trametes lactinea and Trametes cingulata. Our previous and presented studies about 48 basidiomycetes collected in Yemen provide evidence that basidiomycetes from the Arabic region so far should attract more attention as potential source for new biologically active agents.

Biochemical, microbiological and phytochemical studies on aqueous-fermented extracts from Atropa belladonna L. Part 2--Phytochemistry.

Extraction methods of fresh plants into aqueous-fermented extracts according to German Homoeopathic Pharmacopoeia (HAB), regulation nos. 33 and 34 were evaluated. In the course of production, the extraction is accompanied by fermentation and the resulting preparation is stored for at least 6 months until further processing. In part 1 of the work, the biochemical reactions proceeding in aqueous-fermented extracts from the fresh flowering herb of Atropa belladonna var. belladonna (L.) were studied and the responsible microorganisms were identified. This second part aimed at shedding light on the phytochemical changes upon manufacture and storage. Additionally, questions were addressed at the robustness of the method by varying production conditions, its reproducibility and the comparison with ethanolic extraction. Studying 110 extracts produced from 2006 to 2009 as well as model experiments on isolated lactic acid bacteria in atropine solutions proved that the active substance atropine was stable under regular pH conditions. Interestingly, no difference between aqueous-fermented and ethanolic extracts could be found with respect to atropine concentration. In contrast, the amounts of scopoletin and kaempferol glycosides from Atropa belladonna differed depending on the extraction procedure.

Biochemical, microbiological and phytochemical studies on aqueous-fermented extracts from Atropa belladonna L. Part 1--biochemistry and microbiology.

Extraction methods of fresh plants into aqueous-fermented extracts according to German Homoeopathic Pharmacopoeia (HAB), regulation nos. 33 and 34 were evaluated. In the course of production, the extraction is accompanied by fermentation and the resulting preparation is stored for at least 6 months until further processing. The present work aimed at revealing the underlying biochemical reactions during manufacture and storage. In addition, the responsible microorganisms were isolated and identified. To study the robustness of the preparation method, formulation components as well as production conditions were varied. Additionally, questions were addressed at the reproducibility of the method and a comparison with an ethanolic extract was also performed. From 2006 to 2009, 110 extracts from the fresh flowering herb of Atropa belladonna var. belladonna (L.) were produced and analyzed. The results show that lactic acid bacteria (LAB) are primarily involved in the fermentation process, mainly producing lactic acid besides acetic acid and ethanol. The homofermentative Lactobacillus plantarum and the heterofermentative Lactobacillus brevis were identified as predominant lactic acid bacteria. Finally, factors for a successful fermentation are proposed.

Investigation of phenolic leaf extract of Heimia myrtifolia (Lythraceae): Pharmacological properties (stimulation of mineralization of SaOS-2 osteosarcoma cells) and identification of polyphenols.

Evaluation of the activity of an aqueous alcoholic extract obtained from the leaves of Heimia myrtifolia (Lythraceae) by determining its stimulating effect on two human osteoblastic cell lines HOS58 and SaOS-2 indicated its potential for use in the prevention and treatment of osteoporosis. In addition, the extract was found to significantly increase the mineralization of cultivated human bone cell SaOS-2, in which a strong dose-dependent increase was observed. A phytochemical investigation of the extract also confirmed that H. myrtifolia is capable of synthesizing and accumulating appreciable amounts of several phenolics, thus leading to the isolation and characterization of sixteen of these constituents. Identified among these isolates were a new natural product, 1,6-di-O-dehydrotrigalloyl-ß-D-(4)C(1)-glucopyranose, and a rare natural product (this marks its second report), 5,7,4'-trihydroxy-3-methoxyflavanone (dihydrokaempferol-3-O-methyl ether). Structures of these isolates were fully elucidated on the basis of conventional methods of analysis and confirmed by ESI/MS and (1)H and (13)C-NMR analysis.

Aphyllin, the first isoferulic acid glycoside and other phenolics from Tamarix aphylla flowers.

The first glycosylated isoferulic acid, isoferulic acid 3-O-beta-glucopyranoside, together with the new phenolics, tamarixetin 3,3'-di-sodium sulphate and dehydrodigallic acid dimetyl ester have been characterized from a flower extract of Tamarix aphylla. The structures were established on the basis of spectral data. The extract exhibited a distinct radical scavenging effect and to improve the viability of human keratinocytes (HaCaT cells). Also, the known isoferulic acid and ferulic acid which have been determined to be the major components of the investigated extract by HPLC/ESI mass spectrometric screening have been separated, characterized and evaluated as active antioxidants and as cell activity stimulating agents as well.

Evaluation of the in vitro anticancer, antimicrobial and antioxidant activities of some Yemeni plants used in folk medicine.

The present research study deals with the evaluation of sixty four methanolic and aqueous extracts of thirty Yemeni plants used in traditional medicine for their in vitro antiproliferative activity against three human cancer cell lines in a microtiter plate assay based on cellular staining with crystal violet, for their antimicrobial activity against antibiotic susceptible three Gram-positive, three Gram-negative bacterial and one fungal stains and three multiresistant Staphylococcus strains by the agar diffusion method and the determination of MIC against three Gram-positive bacteria with the broth micro-dilution assay, as well as for their antioxidant activity using the DPPH radical scavenging method. Furthermore the chemical composition of the methanolic extracts was determined by using chromatographic methods. As a result of this work, 12 Yemeni herbs namely Centaurothamus maximus, Costus arabicus, Cupressus sempervirens, Dichrocephala integrifolia, Euphorbia schimperi, Gomphocarpus fruticosus, Kanahia laniflora, Meriandera benghalensis, Pulicaria inuloides, Solanum glabratum, Tarconanthus camphoratus and Vernonia leopoldii demonstrated a noteworthy growth inhibitory effect against all cancer cell lines with IC50 values <50 microg/ml. Pronounced antimicrobial activity was observed only against Gram-positive bacteria among them multiresistant bacteria with inhibition zones >15 mm and MIC values <500 microg/ml, by 9 plants especially Centaurothamus maximus, Cupressus sempervirens, Enicostemma verticillare, Meriandera benghalensis, Nepeta deflersiana, Pulicaria inuloides, Tarconanthus camphoratus, Teucrium yemense and Vernonia leopoldii. Moreover, the methanolic extracts of Cupressus sempervirens, Meriandera benghalensis, Pulicaria inuloides and Rhus retinorrhaea showed a remarkable radical scavenging effect at low concentrations.

Bone mineralization enhancing activity of a methoxyellagic acid glucoside from a Feijoa sellowiana leaf extract.

The capability of an aqueous methanol extract obtained from the leaves of Feijoa sellowiana Berg. on possible prevention and treatment of osteoporosis has been examined by evaluating its stimulating effect on the two human osteoblastic cell lines HOS58 and SaOS-2. The extract was found to increase significantly the mineralization of cultivated human bone cell, whereby a clear increment (15.3 +/- 2.7%) in von Kossa positive area was determined when administering 25 microg/ml leaf extract. A phytochemical investigation of the extract has demonstrated the high phenolic content and led to the isolation and identification of twenty three of them, among which the new 3-methoxyellagic acid 4-O-beta-glucopyranoside was fully identified. All structures were elucidated on the basis of conventional analytical methods and confirmed by FTMS, 1D- and 2D-NMR data. The new compound was found to cause a significant increase of mineralized area at 20 microg/mL, while at lower concentrations the effect was not significant. However, an increase of the number of mineralized spots (nodules) at all tested concentrations of the compound was observed.

Antiviral activity of some plants used in Nepalese traditional medicine.

Methanolic extracts of 41 plant species belonging to 27 families used in the traditional medicine in Nepal have been investigated for in vitro antiviral activity against Herpes simplex virus type 1 (HSV-1) and influenza virus A by dye uptake assay in the systems HSV-1/Vero cells and influenza virus A/MDCK cells. The extracts of Astilbe rivularis, Bergenia ciliata, Cassiope fastigiata and Thymus linearis showed potent anti-herpes viral activity. The extracts of Allium oreoprasum, Androsace strigilosa, Asparagus filicinus, Astilbe rivularis, Bergenia ciliata and Verbascum thapsus exhibited strong anti-influenza viral activity. Only the extracts of A. rivularis and B. ciliata demonstrated remarkable activity against both viruses.

Antibacterial and antiviral naphthazarins from Maharanga bicolor.

Maharanga bicolor, Boraginaceae, is used in the Nepalese ethnomedicine for the treatment of several diseases. In the course of screening investigations the dichloromethane extract of the roots of Maharanga bicolor was found to inhibit the growth of gram positive bacteria. Bio-assay directed fractionation led to the isolation of five active naphthazarins, deoxyalkannin (1), alkannin (2), acetylalkannin (3), alkannin beta-hydroxyisovalerate (4) and alkannin beta-acetoxyisovalerate (5). Compounds 2-5 showed antibacterial activity against multi resistant human pathogenic Staphylococcus and Enterococcus species and 1, 4 and 5 showed antiviral activity against herpes simplex virus type-1.

Study of the anticancer potential of Yemeni plants used in folk medicine.

The present work evaluated the anticancer activity of methanol extracts from 24 plants used in Yemeni traditional medicine. To evaluate the in vitro cytotoxic potency of the investigated extracts, an established microtiter plate assay based on cellular staining with crystal violet was used with 5 human cancer cell lines: two lung cancer (A-427 and LCLC-103H), two urinary bladder carcinoma (5637 and RT-112) and one breast cancer (MCF-7) line. The methanolic extracts of Dendrosicyos socotrana, Withanina aduensis, Withania riebeckii, Dracena cinnabari and Buxus hildebrandtii exhibited the highest toxicity on all tumor cell lines with IC50 values ranging between 0.29 and 5.54 microg/ml. The extracts of Jatropha unicostata and Punica protopunica showed a moderate potency on the most tumor cell lines.

Ericifolin: an eugenol 5-O-galloylglucoside and other phenolics from Melaleuca ericifolia.

Ericifolin, an eugenol 5-O-beta-(6'-O-galloylglucopyranoside) possessing the naturally unknown phenolic moiety, 5-hydroxyeugenol, together with the two new phenolics, 2-O-p-hydroxybenzoyl-6-O-galloyl-(alpha/beta)-4C1-glucopyranose and 3-methoxyellagic acid 4-O-rhamnopyranoside have been isolated from the antibacterial leaves extract of Melaleuca ericifolia. In addition, 19 known phenolics were also separated and characterized. All structures were elucidated on the basis of analysis of 1H, 13C NMR, HMQC, HMBC and FTMS spectral data.

Screening of selected Arabian medicinal plant extracts for inhibitory activity against peptidases.

The methanolic extracts of 20 medicinal plants from the island Soqotra/Yemen were screened with respect to their inhibitory potency against angiotensin converting enzyme (ACE), neutral endopeptidase (NEP) and aminopeptidase N (APN). Eight extracts did not show significant inhibitory activity against the enzymes tested, only Kalanchoe farinacea, Boswellia elongata and Cissus hamaderohensis inhibited all three enzymes. The most active extract was prepared from Kalanchoe farinacea characterized by low IC50 values especially for NEP and APN.

Bioactive components of the traditionally used mushroom Podaxis pistillaris.

In the course of an ethnobotanical study on fungi used in Yemeni ethnomedicine the fungus Podaxis pistillaris (Podaxales, Podaxaceae, Basidiomycetes) was found to exhibit antibacterial activity against Staphylococcus aureus, Micrococcus flavus, Bacillus subtilis, Proteus mirabilis, Serratia marcescens and Escherichia coli. In the culture medium of P. pistillaris three epidithiodiketopiperazines were identified by activity-guided isolation. Based on spectral data (NMR, ESI-MS and DCI-MS) their identity was established as epicorazine A (1), epicorazine B (2) and epicorazine C (3, antibiotic F 3822), which have not been reported as constituents of P. pistillaris previously. It is assumed that the identified compounds contribute to the antibacterial activity of the extract.

Phenolics from extracts of Brahea armata with inhibitory effect against 5alpha-reductase type-II.

Fractions of the aqueous alcohol extracts of the rind and kernel of Brahea aramata fruits have been investigated for their activity against 5alpha-reductase type II, which is expressed predominantly in the prostate. This isozyme represents a major target for drugs against benign prostate hyperplasia (BPH) and prostate cancer. Also, a structural analysis of the phytophenolics, present in both aqueous alcohol extracts as the major constituents, has led to the isolation of five phenolics, including the new natural product, 4',6'-dimethoxy beta,4,2'-trihydroxy chalcone from the rind extract and three phenolics, including the new natural product, 1-p-hydroxybenzoyl glycerol from the kernel extract. All structures were confirmed by ESI-MS and NMR analysis.

Red clover extracts stimulate differentiation of human osteoblastic osteosarcoma HOS58 cells.

Different extracts from red clover (Trifolium pratense L.) were tested for their ability to stimulate the activity of osteoblastic osteosarcoma cells (HOS58). As a key marker of osteoblasticity we chose the activity of alkaline phosphatase (ALP). Whereas butanol and methanol extracts had no influence on either ALP or cellular protein production, enzyme activity was increased significantly on incubation with chloroform extracts. All extracts were analysed for isoflavone content. The data clearly suggest a role for red clover isoflavonoids in the stimulation of osteoblastic cell activity.